Fluorescein is an orange-red compound, C20H12O5, that exhibits intense fluorescence in alkaline solution and is used in applications such as medicine for diagnostic purposes, in oceanography as a tracer, and as a textile dye.
Fluorescein was first synthesized by German chemist Adolf Von Baeyer in 1871, from the petroleum derivatives resorcinol (1,3-dihydroxybenzene) and phthalic anhydride. Paul Erlich, a German bacteriologist, employed the fluorescent dye (as sodium salt fluorescein), then known as “uranin”, to track the pathway of secretion of aqueous humor in the eye. This is said to be the first instance of fluorescent dye use in vivo to study physiology.
Fluorescein angiography is an important diagnostic tool that permits study of the condition of the blood vessels of the back of the eye. These vessels are a factor in many diseases that involve the retina. Angiography is performed by injecting fluorescein into a vein in the subject's arm. Within a short time (i.e., typically from a few to several seconds), the dye travels to the vessels in the back of the eye, and a camera with special filters is employed to image the dye as it circulates in the ocular blood vessels. Through examination of the images so produced, an assessment can be made about any circulation problems, for example, vessel leakage, swelling, abnormal or new vessels, and so on.
Fluorescein absorbs blue light, with peak absorption and excitation occurring at wavelengths between 465-490 nm. Fluorescence occurs at the yellow-green wavelengths of 520-530 nm. Although commonly referred to as fluorescein, the dye used in angiography is fluorescein sodium, the soluble disodium salt of fluorescein.
The normal adult dosage of fluorescein is 500 mg injected intravenously. It is typically packaged in doses of 5 mL of a 10% solution or 2 mL of a 25% solution. Upon entering the circulatory system, approximately 80% of the dye molecules bind to serum protein. The remaining unbound or free fluorescein molecules fluoresce when excited with light of the appropriate wavelength. The dye is metabolized by the liver to form fluorescein monoglucuronide, and is ultimately eliminated through the urine within 24 to 36 hours subsequent to administration.
It has been reported that the purity of fluorescein in fluorescein formulations may be correlated to side effects and tolerance to injection. (“Effective differences in the formulation of intravenous fluorescein and related side effects” by Yannuzi et al. in Am. J. Ophthalmol. 1974, 78 (2) pages 217-221). The elimination of all or substantially all impurities from fluorescein compositions utilized for angiography is therefore a primary objective of the present invention.
The following publications may be referred to for additional information regarding fluorescein compositions and processes for preparing and purifying fluorescein.
German Patent No. 136498 (Friedrich et al.) entitled “Process for Preparing Highly Purified Fluorescein for Injection Purposes” describes a process for preparing fluorescein using pyridine.
U.S. Patent Application Publication No. US2006/0106234A1 (Tran-Guyon et al.), entitled “High Purity Phthalein Derivatives and Method for Preparing Same”, describes a process for preparing fluorescein using an anhydrous solvent.
The following patents or publications may also be consulted for further background: U.S. Pat. No. 5,637,733 (Sujeeth), entitled “Synthesis of Fluorescein Compounds with Excess Resorcinol as a Solvent” and U.S. Pat. No. 1,965,842 (Kranz) entitled “Production of Hydroxybenzene-Phthaleins”.
Highly purified fluorescein is necessary for the preparation of solutions for injection purposes. The purified fluorescein used should ideally be: (i) free from impurities, which may be toxic and/or lack fluorescence; (ii) low in salt, which can lead to an unacceptably high osmolality or hypertonicity of the injectable fluorescein product; and (iii) low in color. Certain impurities are strongly colored. Absence of color at particular frequencies may therefore indicate the absence of such impurities. The color profile of fluorescein compositions is therefore considered a significant quality attribute and a visual marker of purity.
A method is needed to identify and quantify very low levels of impurities that may be present in fluorescein compositions. Such a method should be able to separate, identify, and quantify those impurities which may be present.
Thus, there is a need for a fluorescein composition that is highly pure, low in color, low in sodium chloride content and a process to produce such fluorescein that does not require the use of pyridine or other non-aqueous (and potentially noxious) solvent, as well as a method for determining the purity of such fluorescein. The present invention is directed to satisfying this need.